The role of glycine in regulated cell death.

The cytoprotective effects of glycine against cell death have been recognized for over 28 years. They are expressed in multiple cell types and injury settings that lead to necrosis, but are still not widely appreciated or considered in the conceptualization of cell death pathways. In this paper, we review the available data on the expression of this phenomenon, its relationship to major pathophysiologic pathways that lead to cell death and immunomodulatory effects, the hypothesis that it involves suppression by glycine of the development of a hydrophilic death channel of molecular dimensions in the plasma membrane, and evidence for its impact on disease processes in vivo.

Enhancement of glomerular immune complex deposition by a circulating polycation.

It is known that polycations bind to and neutralize glomerular polyanions. Here we examine the effect of the polycation polyethyleneimine (PEI) on glomerular deposition of preformed immune complexes. Bovine serum albumin (BSA)-anti-BSA immune complexes made in 40 times antigen excess were administered following intravenous injection of PEI. Glomerular localization of immune deposits was assessed by quantitative immunofluorescence and electron microscopy and compared to controls receiving diluent without PEI followed by the same dose in immune complexes. In rats receiving PEI, deposits were localized within the glomerular basement membrane (GBM) of all peripheral capillary walls and in the mesangium. In controls, deposits localized exclusively within the mesangium in smaller amounts than after PEI. Thus, neutralization of glomerular polyanion by a circulating polycation enhances the deposition and alters the distribution of immune complexes in glomeruli.

An ultrastructural study of glomerular permeability in aminonucleoside nephrosis using catalase as a tracer protein.

Beef liver catalase (mol wt 240,000) was injected intravenously into normal rats and rats made nephrotic with aminonucleoside of puromycin. The localization of the tracer in the kidneys was then studied by ultrastructural cytochemistry, 3 min-12 hr after injection. Passage of catalase into the urinary space in normal rats was restricted by the basement membrane and by the epithelial slit pore. Nephrotic glomeruli showed extensive fusion of foot processes and formation of pockets and vacuoles in the fused epithelium; within 3 min after injection, catalase appeared in basal pockets, epithelial vacuoles, and the urinary space. Residual slit pores and close junctions in fused epithelium were impermeable to catalase. These studies indicate that alteration of the epithelial cells and basement membrane is responsible for protein leakage in aminonucleoside nephrosis.

Glomerular permeability. Ultrastructural studies in experimental nephrosis using horseradish peroxidase as a tracer.

Wistar/Furth rats were made nephrotic by daily administration of amino-nucleoside of puromycin, and the ultrastructural localization of horseradish peroxidase (mol wt 40,000) in the renal glomerulus was studied from 1 min to 20 hr after intravenous injection of the tracer. In control rats, peroxidase permeated the endothelial fenestrae, the basement membrane, and the epithelial slits, and was present in tubular lumina. Nephrotic glomeruli showed relatively normal basement membranes, extensive fusion of foot processes with formation of "close" intercellular junctions, and large vacuoles and pockets in epithelial cells. On serial sections some of the epithelial vacuoles communicated on one side with the extracellular space overlying basement membrane, and on the other side with the urinary space. In nephrotic animals, peroxidase permeated the basement membrane and the close junctions, and was present in many of the vacuoles and pockets as early as 1 min after injection. Only small numbers of peroxidase-positive vacuoles remained in. epithelial cells 1 hr or more after injection of the tracer. It is suggested that the epithelial pockets and vacuoles form pathways across which leaking proteins can be transferred across the epithelium into the urinary space. Epithelial vacuoles may also be absorption droplets designed to "conserve" leaking proteins, but this function was not prominent in our experiments with peroxidase.

An ultrastructural study of glomerular permeability using catalase and peroxidase as tracer proteins.

Mice were injected intravenously with beef liver catalase (mol wt 240,000) and very small doses of horseradish peroxidase (mol wt 40,000) and the site of localization of these enzymes in the kidney was studied by ultrastructural cytochemistry. 1 min after injection, catalase was present in glomerular capillary lumina and there was minimal permeation of the basement membrane. After 5-180 min, staining of the basement membrane increased progressively but was usually less than that in capillary lumina. At all time intervals the inner (sub-endothelial) layer of the basement membrane contained more reaction product than the lamina densa and the outer (subepithelial) layer. Catalase permeated the entire thickness of the basement membrane and extended up to the slit pore but not beyond the level of the slit diaphragm and was not seen in the urinary space or tubular lumina. Horseradish peroxidase permeated the whole thickness of the basement membrane within 2 min after injection; however, gradients of staining from the inner to outer layers of the basement membrane were frequently seen. The findings with both enzymes indicate that (a) the basement membrane restricts the passage of proteins over a wide range of molecular size with increasing impediment for larger molecules and (b) the slit pore functions as an additional barrier for molecules that cross the basement membrane.

Regulation of the mitochondrial permeability transition in kidney proximal tubules and its alteration during hypoxia-reoxygenation.

Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg(2+). To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg(2+) greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg(2+). Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg(2+), or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

The use of beef liver catalase as a protein tracer for electron microscopy.

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.

Role of molecular charge in glomerular permeability. Tracer studies with cationized ferritins.

Mouse kidneys were perfused with Krebs-Ringer bicarbonate buffer (KRB) containing native, anionic horse spleen ferritin or various cationized derivatives, and the glomerular localization of the probe molecules determined by electron microscopy. Ferritins cationic with respect to the medium (KRB, pH 7.45) accumulated in the subendothelial layers of the glomerular basement membrane (GBM) in amounts far exceeding those observed with anionic ferritins, the degree being greater for the more cationized derivatives. Strongly cationized ferritins, in addition permeated the full thickness of the GBM in considerable amounts, but appeared to be retarded from entry into the urinary spaces at the level of the filtration slits. Very strongly cationized derivatives adhered to glomerular endothelium and GBM and formed aggregates in the outer layers of the latter. The results suggest that intrinsic negative charges are present in the GBM and endothelium, and that the barrier function of the glomerular capillary wall may be ascribed in part to its electrophysical properties.

Glomerular epithelium: structural alterations induced by polycations.

Perfusion of rat kidneys with the polycation protamine sulfate caused glomerular epithelial alterations resembling those observed in human and experimental nephrotic states. The changes included swelling, blunting, and flattening of epithelial foot processes, were accompanied by decreased stainability of glomerular anionic sites, and were largely reversed by subsequent perfusion with the polyanion heparin.

Glomerular permeability of macromolecules. Effect of molecular configuration on the fractional clearance of uncharged dextran and neutral horseradish peroxidase in the rat.

Molecular parameters other than size and charge are likely to influence the filtration of macromolecules across the glomerular filter. We have studies, therefore, the glomerular permeability of macromolecules with widely different molecular configuration such as horseradish peroxidase, a plant glycoprotein with an isoelectric point in the physiologic pH range, and dextran, an uncharged sugar polymer of D-glucopyranose. Simultaneous fractional clearances were determined for both test macromolecules in five Wistar-Furth rats. The results indicate that for a molecular radius of 28.45 A, as measured by gel filtration, the sugar polymer has a fractional clearance of 0.483 on the average, exceeding that of the protein tracer, with a value of 0.068, by a factor less than 7. We conclude that other molecular parameters such as shape, flexibility, and deformability play important roles in the transport of macromolecules across the extracellular matrix that constitutes the glomerular filter.

Cytosolic-free calcium increases to greater than 100 micromolar in ATP-depleted proximal tubules.

Previous studies have shown that cytosolic-free Ca2+ (Caf) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca2+-sensitive fluorophore, fura-2, because of its high affinity for Ca2+. Now, we have used two low affinity Ca2+ fluorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the full magnitude of Caf increase. Between 30 and 60 min after treatment with antimycin to deplete ATP in the presence of glycine to prevent lytic plasma membrane damage, Caf measured with mag-fura-2 exceeded 10 microM in 91% of tubules studied and 68% had increases to greater than 100 microM. Caf increases of similar magnitude that were dependent on influx of medium Ca2+ were also seen using the new low Ca2+ affinity, Mg2+-insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypoxia, and these increases were reversed by reoxygenation. Total cell Ca2+ levels in antimycin-treated or hypoxic tubules did not change, suggesting that mitochondria were not buffering the increased Caf during ATP depletion. Considered in the context of the high degree of structural preservation of glycine-treated tubule cells during ATP depletion and the commonly assumed Ca2+ requirements for phospholipid hydrolysis, actin disassembly, and Ca2+-mediated structural damage, the remarkable elevations of Caf demonstrated here suggest an unexpected resistance to the deleterious effects of increased Caf during energy deprivation in the presence of glycine.

Role of increased cytosolic free calcium in the pathogenesis of rabbit proximal tubule cell injury and protection by glycine or acidosis.

To assess the role of increased cytosolic free calcium (Caf) in the pathogenesis of acute proximal tubule cell injury and the protection afforded by exposure to reduced medium pH or treatment with glycine, fura-2-loaded tubules were studied in suspension and singly in a superfusion system. The Ca2+ ionophore, ionomycin, increased Caf to micromolar levels and rapidly produced lethal cell injury as indicated by loss of lactate dehydrogenase to the medium by suspended tubules and accelerated leak of fura and failure to exclude Trypan blue by superfused tubules. Decreasing medium Ca2+ to 100 nM prevented the ionomycin-induced increases of Caf and the injury. Reducing medium pH from 7.4 to 6.9 or adding 2 mM glycine to the medium also prevented the cell death, but did not prevent the increase of Caf to micromolar levels. Cells treated with 1799, an uncoupler of oxidative phosphorylation which produced severe adenosine triphosphate (ATP) depletion, did not develop increases of Caf until just before loss of viability. Preventing these increases of Caf with 100 nM Ca2+ medium did not protect 1799-treated cells. Reduced pH and glycine protected 1799-treated cells without ameliorating the increases of Caf. These data demonstrate the toxic potential of increased Caf in the proximal tubule and show that Caf does sharply increase prior to loss of viability in an ATP depletion model of injury, but this increase does not necessarily contribute to the outcome. The potent protective actions of decreased pH and glycine allow the cells to sustain increases of Caf to micromolar levels in spite of severe, accompanying cellular ATP depletion without developing lethal cell injury.

Energy thresholds that determine membrane integrity and injury in a renal epithelial cell line (LLC-PK1). Relationships to phospholipid degradation and unesterified fatty acid accumulation.

This study related ATP levels with membrane damage, lipid abnormalities, and cell death in energy-depleted LLC-PK1 cells. Oxidative phosphorylation was inhibited by antimycin A, and glycolysis was regulated by graded glucose deprivation to achieve stepwise ATP depletion. Over a range of ATP levels down to approximately equal to 5% of normal, over 5 h, cells were altered only minimally, or injured reversibly. Such cells maintained mitochondrial potential, and retained more K+ than cells without an energy source. Over the same duration, cells without an energy source were lethally injured. Treatment with antimycin induced increments of triglycerides and decreases of phospholipids. With severe ATP depletion (approximately equal to 5-10% of normal after 5 h), decrease of phospholipids was marked. Cells in which ATP was not measurable (or was less than 5% of normal) showed comparable phospholipid declines but, in addition, showed massive and progressive increase of unesterified fatty acids. The results identified a low threshold of ATP, at best 5-10% of normal, which preserved viability in LLC-PK1 cells despite major loss of membrane phospholipids. This threshold also determined the ability of cells to maintain their normally low levels of unesterified fatty acids. Failure of energy-dependent mechanisms that normally metabolize unesterified fatty acids may be a correlate of the extent of energy depletion that determines lethal injury.

Mechanisms of cell death in hypoxia/reoxygenation injury.

Investigation of death pathways during cell injury in vivo caused by ischemia and reperfusion is of clinical importance, but technically difficult. Heterogeneity of cell types, differences between organ systems, diversity of death paradigms and exacerbation of tissue damage caused by inflammation are only some of the variables that need to be taken into account. With respect to the identification of necrosis and apoptosis in affected organs, technical issues related to preparation artifacts, occurrence of internucleosomal DNA cleavage in necrotic as well as apoptotic cells and other overlaps in death pathways bear consideration. In that caspase independent as well as caspase dependent processes cause cell death and that caspase inhibitors can act as anti-inflammatory agents, evaluation of ischemic death mechanisms in parenchymal cells needs to be performed with caution. When the effects of inflammation are removed by appropriate in vitro studies using purified or cultured cells, several mitochondrial factors that lead to cell death can be studied. Substantial evidence exists for the participation of electron transport defects, mitochondrial permeability transitions (MPT) and release of cytochrome c from mitochondria, effected by pro-apoptotic proteins such as Bax. The anti-apoptotic protein Bcl-2 exerts an overriding protective role in this type of injury by preserving mitochondrial structure and function. In contrast, caspase inhibitors cannot offer long-term protection to ischemically injured parenchymal cells regardless of how effectively they can inhibit apoptotic events, if the cells have suffered permanent mitochondrial damage impairing respiration.

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.

We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

Kidney complications.

Diabetic glomerulosclerosis in man and in all spontaneous-onset and chemically induced diabetes in experimental models is characterized by diffuse increase in mesangial matrix and glomerular basement membrane thickening. The most prominent features of the biochemical changes in the glomerular basement membrane are increase in the collagen-like components, decreased sialic acid, and increased glucosylation. However, the heterogeneity of the various glycoprotein components of the glomerular basement membrane and related components of the mesangium make comparative biochemistry difficult. Increased glomerular blood flow with no apparent alterations in the glomerular filtration coefficient in diabetes may be attributed to altered vascular control mechanisms which may include both hormonal mediation as well as changes in end-organ responsiveness. Although proteinuria is a common manifestation of diabetic involvement of the glomerulus, there is little biochemical or physiologic evidence as to the specific causes of increased glomerular filtration apparatus permeability. Further information as to the pathogenesis of diabetic vascular disease of the kidney and the ability to reverse pathologic changes by correction of the metabolic milieu will require analysis of carefully selected animal models. Particular care in experimental design must include the ability to integrate pathology, physiology, and biochemistry in each model in order to relate the information to human renal diabetic complications.

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.

Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death.

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.

PI3K regulation of the SKP-2/p27 axis through mTORC2.

The cyclin-dependent kinase inhibitor p27 is a key regulator of cell-cycle progression. Its expression and localization are altered in several types of malignancies, which has prognostic significance in cancers such as renal cell carcinoma (RCC). S-phase kinase-associated protein 2 (SKP-2) is an F-box protein that is part of the SKP-1/Cul1/F-box ubiquitin ligase complex that targets nuclear p27 among many other cell-cycle proteins for proteosomal degradation. Its overexpression has been observed in several tumor types. Signaling by phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) has previously been shown to regulate the SKP-2/p27 axis. Recent evidence suggests that PI3K signaling may activate mammalian target of rapamycin complex 2 (mTORC2) activity. As PI3K signaling is known to regulate SKP-2 and p27, we sought to determine whether these effects were mediated by mTORC2. Here we provide additional genetic evidence that PI3K signaling activates mTORC2 kinase activity. We also demonstrate a novel role for mTORC2 in the modulation of nuclear p27 levels. In particular, mTORC2 signaling promotes the reduction of nuclear p27 protein levels through the increased protein expression of SKP-2. These are the first data to demonstrate a role for mTOR in the regulation of SKP-2. In concordance with these findings, mTORC2 activity promotes cell proliferation of RCC cells at the G1-S interphase of the cell cycle. Collectively, these data implicate mTORC2 signaling in the regulation of the SKP-2/p27 axis, a signaling node commonly altered in cancer.